We are developing a gene targeting program based on oligonucleotides that form stable triple helix complexes with specific sequences in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. We have prepared TFOs containing novel sugar analogues and have identified a modification format that supports efficient targeting of specific chrosomal sequences in living mammalian cells. In our developmental work we prepared TFOs, linked to a photoactive DNA mutagen, directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. Treatment of S phase cells with these TFOs resulted in targeted crosslinking in 30% of the cells and 5-10% mutation frequencies. Both crosslinking and mutagenesis were much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. This strategy has been extended to other genes for which genetic analyses of targeting are not possible. For example, we have recently shown, using a biochemical assay, that a site in the human beta globin gene can be targeted at high efficiency. We also find that the targeting oligonucleotides can be used to direct homologous recombination and gene conversion. Cells treated with the TFO and a donor DNA with homology to the region around the target sequence show knock-in of the donor DNA at frequencies 3-4 orders of magnitude above that seen with cells treated with donor alone. We have studied the genetic requirements for repair and mutagenesis of the targeted crosslinks. We find a requirement for the ERCC1/XPF complex for targeted base substitutions. In contrast the appearance of deletion mutations is independent of all nucleotide excision repair, double strand break repair, and recombinational repair genes.[unreadable] We have synthesized antigen tagged photoactive DNA crosslinkers and have shown that the reaction with genomic DNA in living cells can be confined to a discrete nuclear region by photoactivation of that region with a UV laser. We are now studying the repair of the localized crosslinks in repair proficient and deficient cells.